Micro-Raman spectroscopy was used to investigate the chemical composition, microstructure and crystalline phase of an iron oxide sample with three-layer macro-structure prepared by iron oxidation. Two laser lines of 514 and 633 nm with a power of 0.5 mW on the sample were employed to excite the Raman spectra. Comparing the sample spectra to that of bulk alpha-Fe2O3, the sample Raman peaks were assigned. And it was found for the top-layer that the Raman frequencies were down shifted and the peak widths broadened. Therefore we verified that the top-layer of the verified sample is a nano-structure alpha-Fe2O3, the main component of the middle-layer is Fe3O4 and the bottom-layer is most like bulk alpha-Fe2O3.